The present invention is a method for reproducing coniferous plants by somatic embryogenesis using the techniques of plant tissue culture. It is especially suited for producing large clones of superior selections useful for reforestation.
Loblolly pine (Pinus taeda L.), its closely related southern pines, and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) are probably the most important commercial species of temperate North American timber trees. Similarly, Norway spruce (Picea abies (L.) Karst.) is probably the most important European softwood species. Since the early 1940s, when serious private reforestation efforts began, literally billions of one and two year old nursery-grown trees have been planted on cutover or burned forest lands. For many years these seedling trees were grown using naturally produced seed from cones collected as a part time effort of individuals seeking to supplement their incomes. As early as 1957 forest geneticists began to plant seed orchards using either seed or grafted scions obtained from superior trees discovered in the forests. These trees were selected for such inheritable characteristics as rapid growth, straightness of bole, wood density, etc. Now in both the southern pine and Douglas-fir regions the bulk of the seed is produced from selected trees grown in seed orchards, some of them now second and third generation orchards.
Despite the fact that the orchards were stocked with superior trees, pollination often cannot be carefully controlled and frequently the seed trees are fertilized by wild pollen of unknown characteristics. For this reason, the characteristics of the progeny produced by sexual reproduction have not been as predictable as hoped and genetic gain could not be attained as rapidly as desired.
Beginning about 1960, techniques were developed for reproducing some species of plants by tissue culture. These were predominately angiosperms and usually ornamental house plants. The method employed use of a suitable explant or donor tissue from a desirable plant. This was placed on a series of culture media in which nutrients and growth hormones were carefully controlled from step to step. The usual progression was growth from the explant to a callus. The callus was placed on a budding medium where adventitious buds formed. These, in turn, were separated, elongated, and rooted to ultimately form plantlets. A plantlet has the nature of a seedling but is genetically identical to the explant donor plant.
Gymnosperms in general, and most forest tree species in particular, proved to be much more difficult to reproduce by tissue culture. It was not until about 1975 that Douglas-fir was successfully reproduced by organogenesis. Loblolly pine was successfully reproduced about two years later.
Culture by organogenesis is tedious and expensive due to the large amount of delicate manual handling necessary. It was soon recognized that embryogenesis was potentially a much more desirable method from the standpoints of quantity of plantlets produced, cost, and potential genetic gain. Work on embryogenesis of forest species began in the late 1970s. U.S. Pat. No. 4,217,730 to El-Nil describes one early attempt at somatic embryogenesis of Douglas-fir. This approach was later set aside because advanced stage embryos and plantlets could not be readily obtained. However, other workers entered the field in increasing numbers and progress has been rapid even if it has not until the present time reached the commercial stage. A brief review of some of the most important work will follow. This is intended to be representative and is not fully inclusive of all the work in the field. Literature citations in the text are given in abbreviated form. Reference should be made to the bibliography at the end of the specification for full citations of the literature noted herein.
The natural embryogeny of gymnosperms is described in great detail by Singh (1978). Conifer-type embryogeny is one of four types noted for gymnosperms. This includes virtually all of the important forest species except Sequoia. Singh notes that the immature seeds typically contain more than one embryo. Most commonly this seems to occur when a single zygote forms multiple embryos, a phenomenon called "cleavage polyembryony". As the seed matures one embryo becomes dominant while the others are suppressed. The ability to form multiple embryos from a single zygote forms the basis for most of the present embryogenic processes for multiplying conifers. However, Douglas-fir is an exception. Most typically only a single embryo will be present throughout the formation and maturation of a seed. This may account for at least some of the difficulty experienced to date in multiplying Douglas-fir by somatic embryogenesis.
Bourgkard and Favre (1988) describe what is the apparently successful production of plantlets by somatic embryogenesis of Sequoia sempervirens. As a historic note, this was one of the first forest tree species successfully reproduced by organogenesis.
Hakman and her coworkers have concentrated on Norway spruce (Picea abies), apparently with some success. In a paper by Hakman, Fowke, von Arnold, and Eriksson (1985) the authors describe the production of "embryos" but not plantlets. Hakman and von Arnold (1985) do suggest that they have successfully obtained plantlets. This latter paper is interesting for its comments on the variability within the species and the poor success with many of the seed sources used for explants. The authors suggest that this variability may be due to the physiological condition of the source material. However, other workers have noted great differences in behavior between recognized genotypes of the species.
Nagmani and Bonga (1985) describe embryogenesis from megagametophytes of Larix decidua by tissue culture. The archegonia, proembryos, or embryos with their suspensors were removed prior to culture. Some of the resulting embryos produced in culture were stated to have further advanced to become plantlets established in soil. The ploidy of these plants was not investigated.
Successful production of small quantities of plantlets has now been reported for loblolly pine. Teasdale, Dawson, and Woolhouse (1986) showed the criticality of proper mineral nutrients for cell suspension cultures of loblolly pine. The article by Becwar, Wann, and Nagmani (1988) is enlightening for the differences shown in performance between different families (or genotypes). Three families out of the ten tried accounted for most of their success. Even so, they appeared unable to grow cotyledonary embryos. A companion paper by Nagmani and Becwar (1988) showed development of Pinus taeda to the precotyledonary stage. In an earlier paper, Gupta and Durzan (1987) described their success in taking loblolly pine to the plantlet stage by embryogenesis. However, only one genotype was successfully taken to the plantlet stage and only one converted plant was produced. The authors note the need for "improved conversion rates" as well as other information before the process can be considered commercially practical.
Sugar pine (Pinus lambertiana) has also been cultured to the plantlet stage as reported by Gupta and Durzan (1986a). The authors note a very low 1-2% conversion of embryos into plantlets.
The researchers just noted appear to be the only others who have previously achieved success in producing Douglas-fir plantlets by embryogenesis (Durzan and Gupta 1987). Again, the success ratio appears to be very low and they have obtained only two converted plants from a single genotype.
In our earlier application, Ser. No. 321,035, filed Mar. 9, 1989, now U.S. Pat. No. 4,957,866, which is a grandparent to the present application, we described an improved method for reproducing coniferous species by somatic embryogenesis. An intermediate high osmoticant culture medium was used to generate strong late stage proembryos. This was done prior to the development of cotyledonary embryos in a medium containing abscisic acid. The methods disclosed were of particular effectiveness in somatic polyembryogenesis of loblolly pine. In Ser. No. 426,331, filed Oct. 23, 1989, Now U.S. Pat. No. 5,034,326, the other grandparent application to the present one, we disclosed the use of a combination of abscisic acid with activated charcoal in a cotyledonary embryo development medium. The charcoal is believed to gradually reduce the concentration of abscisic acid during the development period. This improvement resulted in the production of more robust embryos with a much reduced tendency for precocious germination.
Activated charcoal has been widely used before in tissue culture media where it is believed to function as an adsorbent for toxic metabolic products and undesirable amounts of residual hormones. Abscisic acid has also been recognized as being a useful plant hormone in cultures inducing conifer embryogenesis; e.g., Boulay, Gupta, Krogstrup, and Durzan (1988). The combination of these two materials has been used by a number of workers, generally with indifferent or negative results. Johansson, Andersson, and Ericksson (1982) cultured anthers of several ornamental plant species using a two phase liquid over solid medium in which the agarified solid phase contained activated charcoal. The charcoal appeared to be useful for absorbing small amounts of endogenous abscisic acid. In a related paper, Johansson (1983) tested the effects of charcoal as an adsorbent of materials inhibiting the initiation of embryogenesis. In a test intended as a model, he added exogenous ABA in amounts varying by orders of magnitude between 10.sup.-9 M and 10.sup.-3 M to media with and without activated charcoal in the solid portion of a two phase medium. His conclusion was that initiation was completely inhibited for all of the test species at ABA concentrations above 10.sup.-6 M, when no charcoal was used, and 10.sup.-4 M when charcoal was present. Thus, charcoal was seen as an effective material for removing inhibitory amounts of ABA and other undesirable materials such as phenolics.
Ziv and Gadasi (1986) studied embryogenesis in several genotypes of cucumber (Cucumis sativus L.). They used liquid cultures as well as the two layer technique with activated charcoal in the solid layer of the medium and low (0.4 .mu.M) levels of abscisic acid in the liquid layer. In the liquid cultures abscisic acid by itself only slightly improved embryo formation and was significantly more effective than the combination of abscisic acid with activated charcoal. Plantlet development in the liquid over solid cultures was slightly improved by the combination of the two materials.
Buchheim, Colburn, and Ranch (1989) suggest that exogenous abscisic acid and activated charcoal would probably not be a very useful combination of ingredients in a culture medium because of adsorption of the abscisic acid by the charcoal with subsequent loss of its biological effectiveness.
Since the importance of the osmotic environment within a developing seed is known (Yeung and Brown 1982), it has been assumed by others that the osmotic potential of the media during a culturing process could have an important effect (e.g., Raghavan 1986). Lu and Thorpe (1987), using white spruce (Picea glauca), noted that increasing the osmolality of a medium and reducing the auxin concentration enhanced development and maturation of somatic embryos. They observed that more embryos developed on media containing 6% than on those with 9% sucrose and that similar results were obtained when sorbitol replaced 3% of the sucrose in the medium. Sorbitol is known to be only poorly metabolized so presumably its effect was osmotic rather than as a carbon source for the developing embryos. Quite in contrast to their findings, Hakman and von Arnold (1988), using the same species and a combination of abscisic acid and sucrose in a development medium, found a very sharp falloff in success in going from 3% to 4% sucrose.
Becwar and Feirer (1989) note work involving the transfer of a loblolly pine embryonal-suspensor mass to development media containing 10 .mu.M abscisic acid with 3-6% sucrose. However, they reported no details of their experimental protocol and only that the media "promoted embryo development". A later paper by Becwar, Nagmani, and Wann (1990) gives further details of initiation of the cultures and describes hormone requirements and the effect of embryo maturity for 10 full sib families.
Finer, Kreibel and Becwar (1989), studying eastern white pine (Pinus strobus L.), initiated and maintained cultures on media with a 3% sucrose level. Further embryo development was then attempted on a medium with 1-12% sucrose combined with a high concentration of abscisic acid and varying amounts of glutamine. Best results were found with 50 mM glutamine, 38 .mu.M abscisic acid, and 6% sucrose. However, the number of embryos formed under any of the conditions was not high and, as of the time of reporting, none has been successfully germinated and converted into plants.
Schuller and Reuther (1989), in the abstract of a paper, describe the study of several sugars and soluble starch as carbohydrate sources for the culture of Abies alba. They note that development was obtained only on a medium using soluble starch and lactose. No details were given and apparently no somatic embryos were developed to the cotyledonary stage.
Von Arnold (1987) investigated carbohydrate level of the initiation medium for Norway spruce. Sucrose was varied between about 1-3% with successful initiation being obtained at the higher level on half strength medium. By replacing a portion of the sucrose with sorbitol she showed that the poorer results on full strength medium were not due to increased osmotic pressure.
Von Arnold and Hakman (1988) took a Norway spruce embryogenic callus and transferred it to a modified intermediate medium prior to full embryo development. The intermediate medium contained abscisic acid and from 1-3% sucrose. The higher sucrose levels, along with the abscisic acid, resulted in increased frequency of advanced stage proembryo development.
The potential for achieving genetic gain using somatic embryogenesis is recognized as being very great. However, the problems to date have been so overwhelming that commercial application has seemed reasonably close at hand only for Norway spruce and, to a lesser extent, loblolly pine and Douglas-fir using the methods described in our parent applications. Possible large scale commercial production of replanting stock by embryogenesis has remained no more than a fond hope in the minds of the people working in the field.